Imatinib Mesylatemodulates Regulators of Quiescence inGastrointestinal Stromal Tumor (GIST) cells

Joshua A. Parry, Matthew F. Brown, Danushka Seneviratne,Stefan Duensing, Anette Duensing

University of Pittsburgh Cancer Institute,Pittsburgh, PA

Clinical and experimental findings suggesttumor cell quiescence in imatinib-treated GISTMajor problems of imatinib therapy: therapy resistance incomplete responses

Detectable tumor mass under treatmentRelapse when imatinib therapy is terminated

Tumor cells that do not respond to imatinib by undergoing apoptosis but remain quiescent may be a reservoir for resistant GIST cellsHoldsworth, Am J Roentgenol 2007;189:W324-W330transgenic KitV558 /+ mouse(in collaboration with C. Antonescu and P. Besmer)PETCTbaselineimatinib

Tumor cell quiescenceDefinition:lack of growth/proliferation, non-dividing stateexit of cell division cycle in G0/G1 reversibleIn contrast to senescence (= irreversible)Molecular regulators:p27Kip1DREAM complex

Problem for cancer therapy:quiescent cells do not respond to anticancer agents that target proliferating cellsquiescent cells are unlikely to undergo apoptosis

Regulation of quiescenceproliferating cellscells in G0/G1APC inactiveSKP2 highp27 lowCDK activeSKP2 lowp27 highCDK inactiveAPC activeDREAM activeDREAM inactiveD R E A MDMREA

Effects of imatinib on regulators of quiescenceGIST882GIST882

p27Kip1 upregulation in mouse xenograftsGIST882xenografts

p27Kip1 and SKP2 - risk for progressionSKP2 (p 63.7% p27 pos. cellsTotalNone, very low, low, intermediate61319 High639Total121628

Risk of progression (NCCN) 5 SKP2 pos. cells/HPF> 5 SKP2 pos. cells/HPFTotalNone, very low, low, intermediate17017 High459Total21526

Non-apoptotic cells enter quiescenceafter imatinib

Quiescent cells can re-enter the cell cycle

Imatinib and the DREAM complexmammalian DREAM complex(DP, RB-like (p130), E2F and MuvB=LIN)E2F target gene

Conclusions imatinib leads to cell cycle exit and cellular quiescence modulation of the APCCDH1 SKP2 p27Kip1 axis modulation of the DREAM complex members

quiescent cells readily re-enter the cell cycle after removal of imatinib

compounds that modulate these pathways as potential antitumor agents in GIST?

AcknowledgmentsDuensing LabJoshua ParryDanushka SeneviratneMatthew BrownJulianne BaronYing LiuSophie PerdreauPayel ChatterjeeStefan Duensing

American Cancer Society(#RSG-08-092-01-CCG)GIST Cancer Research FundLife Raft Group (#UPCC-AD-100108)University of PittsburghShih-Fan Kuan

Brigham & Women's HospitalJonathan Fletcher

Katolieke Universiteit LeuvenMaria Debiec-RychterPatrick Schffski

Dana Farber Cancer InstituteJames DeCaprioLarisa Litovchick

Memorial Sloan KetteringPeter BesmerCristina Antonescu

Pro-apoptotic Activity of Bortezomib in Gastrointestinal Stromal Tumors (GIST) cells

Sebastian Bauer1, Joshua A. Parry2, Thomas Mhlenberg1,Payel Chatterjee2, Shih-Fan Kuan2, Jonathan A. Fletcher3,Stefan Duensing2, Anette Duensing2

1University of Duisburg-Essen, Essen, Germany2University of Pittsburgh Cancer Institute, Pittsburgh, PA3Brigham & Womens Hospital, Boston, MA

IMATINIBKIT activity tumor cell quiescencerisk of relapsewhen taken off imatinib!GISTAPCCDH1 - SKP2 - p27Kip1

DREAM complex

IMATINIBapoptosistumor cell quiescencerisk of relapsewhen taken off imatinib!GISTAPCCDH1 - SKP2 - p27Kip1

DREAM complexKIT activity

apoptosistumor cell quiescencerisk of relapsewhen taken off imatinib!GISTOTHERCOMPOUNDS?APCCDH1 - SKP2 - p27Kip1

DREAM complex

Histone H2AX is upregulated after imatinib treatmentvariant of core histone H2Arandomly incorporated into nucleosomes rapidly phosphorylated at serine 139 in response to genotoxic stress -H2AXmediates DNA damage response reactionspotential tumor suppressorSerine 1391142histone H2AXnucleosome

Upregulation of H2AX is causatively involved in GIST cell apoptosis

H2AX upregulation is due to increasedprotein stabilityIB: ubiquitinUbiquitin/26S proteasome pathwayMani and Gelmann, JCO 2005; 23:4776-4789

Inhibition of the proteasome inducesapoptosis in GISTdose responsetime courseBortezomib:Velcade (Millennium Pharmaceuticals)proteasome inhibitor

FDA-approved for:multiple myelomamantle cell lymphoma

Bortezomib mechanism of action

Bortezomib mechanism of action

Mechanism of action of BortezomibRT-PCRqRT-PCR

Bortezomib inhibits ongoing gene transcription

Bortezomib is effective in imatinib-resistant GIST24 h48 h

Bortezomib is effective in imatinib-resistant GISTGIST004: imatinib-resistant GIST(short-term culture)Kit+/K641E transgenic mice

Inhibition of NF-kB signaling is not involved in Bortezomib-induced apoptosis in GISTbortezomib

Conclusions Bortezomib induces apoptosis in GIST cells

dual mechanism of actionupregulation of H2AXtranscriptional inhibition of KIT

NOT inhibition of NF-kB signaling

new therapeutic option for imatinib-resistant GIST patients

AcknowledgmentsDuensing LabJoshua ParryMatt Brown Dee SeneviratneJulianne BaronPayel ChatterjeeAnna ChinStefan Duensing

American Cancer Society(#RSG-08-092-01-CCG)GIST Cancer Research FundLife Raft Group (UPPCC-AD-100108)Universitt Duisburg-EssenSebastian BauerThomas Mhlenberg

Brigham & Women's HospitalJonathan Fletcher

University of PittsburghShih-Fan Kuan

Cleveland ClinicBrian Rubin

Oregon Health & Science UniversityChristopher CorlessMichael Heinrich

***********but what happens to these cells? can we really mimic what happens in a patient that goes off imatinib? can if they are really quiescent, they should be able to re-enter the cell cycle this is indeed what happens following experiment: treat cells with imatinib for 3 days (most, but not all cells undergo apoptosis) remove imatinib-containing media, replace with regular media follow cells over a period of up to 11 days each day (later every other day) take cells for BrdU and protein extracts what we see is that proliferative activity of cells rapidly declines under imatinib as expected from about 60% to less than 5% on treatment day 3 after removal of imatinib, cells re-enter the cell cycle aproximately after 5 days being in regular media even a rebound effect with a higher percentage of proliferating cells after 9 days, before proliferative index returns to same level as control illustrated here: DMSO control, 3 days imatinib, 9 days after removal of imatinib in parallel, we can see that phosphoKIT immediately rebounds after removal of imatinib levels of SKP2, p27 and cyclin A respond in parallel to what is seen in the BrdU incorporation somewhat delayed

this experiment shows that we are really able to mimic in the tissue culture dish what can possibly happen in a patient who goes off imatinib despite having residual tumor ****************************